Effect of Decalcification Agents on Nucleic Acid Quantity and Quality
Veena M Singh, Ranelle C Salunga, Yen K Tran, Jose Gallindo, Pamela A Plumlee, Sunny W Chu, Mark G Erlander, Michael R Peterson. University of California San Diego, San Diego; bioTheranostics, San Diego
Background: Molecular studies are part of standard care for cancer patients. Bone a common or sole site of metastasis requires decalcification. However commonly used decalcification agents usually contain strong acids that degrade RNA/DNA. We propose to systematically study the effect of a spectrum of commercial decalcifiers (strong acid-hydrochloric/nitric, weak acid-formic and chelating agent-EDTA) on the quantity/quality of RNA/DNA. The paradigm shift in oncology with biomarker targeted therapy and gene expression profiling studies, necessitates improved nucleic acid quality/quantity from bone.
Design: Multiple bone biopsies (obtained from amputations) of similar size and cellularity were fixed in 10% neutral buffered formalin, randomized to various decalcifiers for 2 hours then processed and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA/RNA was quantified in nanograms by PicoGreen and RiboGreen (Life Technologies,Carlsbad,CA) respectively. Assessment of DNA/RNA integrity was accomplished by comparison of the average cycle threshold (CT) by PCR of selected housekeeping genes for each decalcifier. Results were then analyzed by two sample t-test.
Results: There was a significant decrease in both DNA/RNA yield and integrity with strong acids (hydrochloric, nitric) vs. 14% EDTA and formic acid. DNA yield was (mean ng) 5.9 vs. 37.1 (p-value 0.014) and RNA was (mean ng) 39.5 vs. 157.9 (p-value 0.002). DNA integrity was (mean CT) 38.7 vs. 28.9 (p-value 0.00004) and RNA was 34.9 vs. 26.3 (p-value 0.002). A 1-2 CT improvement for 14% EDTA and immunocal was observed.
Conclusions: The decalcification agents associated with the greatest nucleic acid quantity and better quality are 14% EDTA and formic acid decalcifiers. In addition data suggests that RNA integrity improves with 14% EDTA or immunocal.
|DNA Quantity(Picogreen)||DNA Quality (qPCR)Equal ng/reaction||RNA Quantity (Ribogreen)||RNA Quality (qRT-PCR) Equal ng/reaction|
|Commercial Name||Decalcification agent||Yield (ng)||Ave CT||Yield (ng)||Ave CT|
|14% EDTA||14% EDTA||67.8||28.3||226.2||25.19|
|Surgpath decalcifier I||Formaldehyde/Methanol/Formic acid||27.1||29.53||144.8||26.43|
|IMEB HCL/Formic decal solution||HCL/Formic acid||5.8||36.37||50.4||38.8|
|Surgpath decalcifier II||HCL/EDTA||5.3||40.0||30.5||34.8|
|Decal stat||HCL/EDTA||1.4||Quantity Not Sufficient||25.4||Quantity Not Sufficient|